Human hematopoietic activities are carried out primarily in the bone marrow and various kinds of blood cells are produced through a variety of complex pathways. The hematopoietic activities are controlled by specific glycoproteins, which are generally referred to as colony stimulating factors (CSFs). CSFs have been identified and distinguished according to their activities. That is to say, CSFs, which serve as growth factors when blood cells are cultured in semi-solid media, stimulate the clonal formation of monocytes, granulocytes or other hematopoietic cells. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively while multi-CSF, also known as interleukin-3 (IL-3), stimulates the clonal proliferation of various types of blood and tissue cells, such as granulocyte, macrophages, megakaryocytes, red blood cells, or the like.
Specifically, the G-CSF, which is a cytokine of instructing the division and differentiation of intermedullary stem cells and leukocytes outside the bone marrow, has been known to promote the phagocytic activity of neutrophils by stimulating the differentiation and/or proliferation ofneutrophilic progenitor cells and activating mature neutrophils, and the reactivity to chemotactic factors ((Metcalf, Blood) 67:257 (1986); (Yan, et al., Blood) 84(3): 795-799 (1994); (Bensinger, et al., Blood) 81(11):3158-3163 (1993); (Roberts, et al., Expt'l Hematology) 22:1156-1163 (1994); (Neben, et al., Blood) 81(7): 1960-1967 (1993)).
Treatment of malignant tumors is generally performed by radiotherapy and/or chemotherapy, which undesirably result in a sharp reduction of leukocytes and lowered immunity occurring incidental to the reduction of leukocytes, thereby causing significant drawbacks when the therapeutic treatment by radiotherapy and/or chemotherapy is performed over an extended period of time. G-CSFs were initially reported as adjuvants capable of effectively treating the cancer by activating a patient's immunizing capability (Lopez et al., J. Immunol. 131(6):2983-2988, 1983; Platzer et al., J. Exp. Med. 162:1788-1801, 1985), and are currently effectively used for the treatment of a variety of cancers and intractable leukemia.
Research of G-CSFs started initially with the finding that granulocyte-CSF materials are present in the culture medium of human carcinoma CHU-2 cell line (Nomura et al, EMBO J. 8(5):871-876, 1986) or human bladder carcinoma cell line 5637 (Welte et al., Proc. Narl. Acad. Sci. USA82:1526-1530, 1985; Strige et al., Blood 69(5):1508-1523, 1987), and a protein having granulocyte-colony stimulating activity having a molecular weight of 18 to 19 kDa was purified (Nomura et al., EMBO J. 5(5):871-876, 1986).
The cDNAs of G-CSFs were first isolated from human bladder carcinoma cell line 5673 by L. M. Souza et al. (Science, 232: 61-65 (1986) (see Korean Patent Publication No. 1998-77885), and then cloned from cDNA libraries of squamous carcinoma cell line and peripheral blood macrophage (S, Nagata et al., Nature, 319: 415-417 (1986); S, Nagata et al., EMBO J., 5: 575-581 (1986); Y. Komatsu et al., Jpn. J. Cancer Res., 78: 1179-1181 (1987)).
With the advent of gene recombinant technology, it was revealed that G-CSFs are produced as insoluble inclusion bodies in the course of expressing a large amount of G-CSF from Escherichia coli and converted into active G-CSFs by refolding. The recombinant G-CSF (rG-CSF) produced from E. coli comprises a protein identified in SEQ ID NO:2 (175 amino acids), that is, a natural G-CSF (174 amino acids) identified in SEQ ID NO:1 and N-terminal methionine, and has a molecular weight of about 19 kDa.
In addition, the natural G-CSF has an O-glycosylation site in an amino acid threonine (Thr) at position No. 133 while the rG-CSF derived from E. coli has no glycosylation site. However, it is known that the presence or absence of the glycosylation site and/or N-terminal methionine have little effect on the biological activity (Souza et al.: Science 232, 1986, 61).
Biological protein therapeutic agents such as G-CSF have advantageously high selectivity and low toxicity while they have a short in vivo retention time and are unstable. To overcome such drawbacks, there has been developed a method of conjugating biocompatible polymers, e.g., polyethylene glycol (PEG), polyvinyl alcohol (PVA) or polyvinyl pyrrolidone, to biological proteins (polypeptides), e.g., G-CSF or interferon. Such PEG conjugation prevents degradation of biological proteins by effectively inhibiting proteases, increases the stability and half life of a biological protein therapeutic agent by preventing fast excretion of the biological protein therapeutic agent from the kidney, and reducing the immunogenicity (Sada et al. J. Fermentation Bioengineering 71: 137-139 (1991)).
In particular, examples of the PEGylated protein therapeutic agents include a pegylated formulation of adenosine diaminase developed as a therapeutic agent of combined immunodeficiency; a pegylated formulation of interferon developed as a therapeutic agent of hepatitis; pegylated formulations of glucocerebrosidase and hemoglobin, and so on.
In addition to PEG, copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers) are generally used in the chemical coupling of the protein therapeutic agent.
Since the PEG, which is a polymeric compound having a general formula: HO—(—CH2CH2O—)n-H, is highly hydrophilic, it binds with a protein for use in medical applications, thereby increasing the solubility. The PEG binding to the protein has a molecular weight in the range of about 1,000 and about 100,000. If the molecular weight of PEG exceeds 1,000, the PEG has significantly low toxicity. PEGs having a molecular weight in the range of about 1,000 and about 6,000 are present systemically and metabolized through the kidney. In particular, branched PEGs having a molecular weight of about 40,000 are present in organs such as blood, liver, or the like, and metabolized through the kidney.
U.S. Pat. No. 4,179,337 discloses a physiologically active non-immunogenic watersoluble polypeptide composition having polypeptides coupled to polyethylene glycol (PEG) or polypropropylene glycol (PPG) having a molecular weight of 500 to 20,000 daltons. In order to couple the PEG to a polypeptide, an activated PEG is generally used. The activated PEG is prepared by converting one end hydroxy group of the PEG into a methyl ether group and the other end hydroxy group into an electrophilic group, thereby coupling the PEG to the polypeptide through the electrophilic group. However, since such a chemical coupling reaction is non-specific, the PEGylated to the active site of the protein, i.e., the polypeptide, undesirably reduces the activity of the protein, an example of which is found from PEGylated interferon-α, developed by Roche and Schering. A PEG conjugate of the interferon-α developed by Roche and Schering is a conjugate in which a molecule of PEG is combined with a molecule of interferon-α. Although the PEG conjugation increased the in vivo half-life of pegylated interferon-α, PEGs are coupled to various sites of interferon-α, thereby considerably reducing the biological activity.
Examples of commonly used activated PEG include (a) PEG dichlorotriazine, (b) PEG tresylate, (c) PEG succinimidyl carbonate, (d) PEG benzotriazole carbonate, (e) PEG p-nitrophenyl carbonate, (f) PEG trichlorophenyl carbonate, (h) PEG succinimidyl succinate, and the like (M. J. Roberts, M. D. Bentley, J. M. Harris, Chemistry for peptide and protein PEG conjugation, Advanced Drug Delivery Reviews 54 (2002) 459-476). Since the chemical coupling reaction is non-specific, a multimer having multiple PEGs boned thereto or an isomer having PEGs bonded to different sites thereof may be produced. The multimer and isomer reduce the biological activity of the PEGylated product, make accurate pharmacokinetic measurement and purification process of the PEG conjugation difficult.
To overcome these problems, U.S. Pat. No. 5,766,897 and WO 00/42175 disclose a method of selectively coupling PEG to cysteine (Cys) residue of protein using PEG-maleimide. A free cysteine that is not associated with disulfide bonding is necessary to couple the PEG to the protein through a cysteine specific PEG conjugation. In the G-CSF containing 5 cysteines, disulfide bonds are formed between cysteines at positions 36 and 42 (based on natural G-CSF) and between cysteines at positions 64 and 74 (based on natural G-CSF), while cysteine at position 17 is a free cysteine. Formation of the two disulfide bonds between cysteine at position 36 and cysteine at position 42 and between cysteine at position 64 and cysteine at position 74 is critically important in the construction of natural G-CSF and maintenance of the biological activity of G-CSF. Since the biological activity of G-CSF is not seriously affected by substitution of cysteine at position 17, cysteine at position 17, with serine, the construction of natural G-CSF and the biological activity of G-CSF are not be attributable to the substitution of cysteine at position 17 with serine (Winfield et al.: Biochem. J. 256, 1988, 213). However, Piget et al. has reported that cysteine at position 17 may form intermolecular disulfide bonds or intramolecular disulfide bonds according to reaction conditions, thereby impeding refolding and lowering the biological activity and stability of G-CSF (Proc. Natl. Acad. Sci. U.S.A., 83, 1986, 7643).
Meanwhile, it has been reported that substitution of cysteine at position 17 of G-CSF, which is the only free cysteine present in G-CSF (Kyowa Hakko, Tokyo, Japan), with serine causes little change in the biological activity of G-CSF (see Korean Patent Registration No. 10-0114369).
One of exemplary therapeutic agents developed using mutant G-CSF is Neu-up® (Component name: Nartograstim) manufactured by Kyowa Hakko, Tokyo, Japan. Reportedly, the mutant G-CSF showed markedly high activity and a longer in vivo half-life compared to natural G-CSF. However, no clinical benefit of the mutant G-CSF has yet been reported.
A pegylated product of N-terminal specific conjugation between G-CSF and PEG, referred to as PEG-G-CSF, was commercially available by Amgen, Inc. (Neulasta®, pegFilgrastim).
Development of mutant G-CSF for chemical conjugations has been proposed by Maxygen Inc. (PCT/DK2001/00011). According to the proposed publication, however, characteristics of the mutant G-CSF and PEG-G-CSFs produced therefrom are not well described. The mutants can be prepared in various manners. That is to say, there are several thousands to several tens of thousands of possible mutants according to the selection of mutation location, mutation type (insertion, substitution, deletion), mutation extent (1-2 residue˜fragment), and combinations thereof, and their biological properties and physiochemical properties for the respective cases would differ. Apparently, chemical conjugates produced from the mutants would differ.
Generally, preparation of mutants for forming chemical bonds is designed based on the knowledge of their structures. According to Osslund et. al. (U.S. Pat. No. 5,581,476), binding sites are selected based on accessible residues, and a binding site that is not interfered from the chemical binding is selected from a structure in which a G-CSF molecule is connected to a G-CSF receptor to restrict the number of target mutants, thereby selecting mutants to be actually subjected to formation of chemical bonds. However, selecting of the mutation position with the knowledge of the structure in such a manner will be easily contemplated by one skilled in the art.
During addition and insertion associated with mutation, the number of residues added and inserted and the character of the mutation (i.e., the type of residues for mutation) considerably affect the biological activity of the mutant. Interchanging a hydrophobic residue and a hydrophilic residue or interchanging a large number of residues and a small number of residues has a substantial effect on the structure of the mutant. One skilled in the art can easily speculate that such structural effect would lead to a considerable change in the biological activity and stability of G-CSF. Particularly, induction of free cysteine to G-CSF exerts a considerable effect on the stability of protein, as proposed by Freeman M L et al. (Destabilization and denaturation of cellular protein by glutathione depletion, Cell Stress Chaperones. 1997 September; 2 (3):191-8).